Three interrelated aspects of transcription by influenza viral nucleocapsid complexes will be studied in order to obtain a basic understanding of the enzymatic activities, other components in complexes that influence activity and possible modulators of primary transcription. 1) Three enzymatic activities, RNA-dependent RNA polymerase, poly A polymerase and an endonuclease required for synthesis of viral mRNAs are present in complexes. From the protein composition of the complexes it is highly likely that the activities are catalyzed by one or more of the P proteins. To determine whether the enzymes are functionally distinct and separable or exist in a multimeric protein, P proteins isolated from complexes will be separated by column chromatographic methods, the proteins identified by gel electrophoresis and their activities determined in reconstitution experiments with RNP complexes devoid of activity. 2) Evidence for an endogenous primer in complexes which permits the synthesis of polyadenylated RNAs has been obtained. Studies on the nature of this primer by a combination of gel electrophoretic and biochemical techniques are planned. 3) Preliminary results indicate that two proteins, one viral and the other cellular, modulate primary transcription. Their activities will be studied by determining whether complexes are altered and how product formation is affected. A fourth area of the proposal involves the development of cell-free systems capable of viral template cRNA and vRNA synthesis. Molecular probes and electrophoretic techniques will be employed to determine the products formed. Fractionation of the systems will then be carried out to isolate the active components with the eventual goal of determining which viral and possible cellular components are required for synthesis.